scholarly journals Solubilization, Activation, and Insecticidal Activity of Bacillus thuringiensis Serovar thompsoni HD542 Crystal Proteins

2008 ◽  
Vol 74 (23) ◽  
pp. 7145-7151 ◽  
Author(s):  
Samir Naimov ◽  
Rumyana Boncheva ◽  
Rumyana Karlova ◽  
Stefan Dukiandjiev ◽  
Ivan Minkov ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pieris Rapae ◽  
Insect Pest ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cydia Pomonella ◽  
Sodium Dodecyl ◽  
Crystal Proteins ◽  
Stable Product ◽  
Insect Gut

ABSTRACT Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542 in a crystal together with a 40-kDa accompanying protein, is one of a small group of nontypical, less well-studied members of the Cry family of insecticidal proteins and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this paper, we describe the characterization of the Cry15Aa and 40-kDa protein's biochemical and insecticidal properties and the mode of action. Both proteins were solubilized above pH 10 in vitro. Incubation of solubilized crystal proteins with trypsin or insect midgut extracts rapidly processed the 40-kDa protein to fragments too small to be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas the Cry15 protein yielded a stable product of approximately 30 kDa. Protein N-terminal sequencing showed that Cry15 processing occurs exclusively at the C-terminal end. Cry15 protein showed in vitro hemolytic activity, which was greatly enhanced by preincubation with trypsin or insect gut extract. Larvae of the lepidopteran insects Manduca sexta, Cydia pomonella, and Pieris rapae were susceptible to crystals, and presolubilization of the crystals enhanced activity to P. rapae. Activity for all three species was enhanced by preincubation with trypsin. Larvae of Helicoverpa armigera and Spodoptera exigua were relatively insensitive to crystals, and activity against these insects was not enhanced by prior solubilization or trypsin treatment. The 40-kDa crystal protein showed no activity in the insects tested, nor did its addition or coexpression in Escherichia coli increase the activity of Cry15 in insecticidal and hemolytic assays.

scholarly journals Genetic Variability of Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) Populations from Latin America Is Associated with Variations in Susceptibility to Bacillus thuringiensis Cry Toxins

2006 ◽  
Vol 72 (11) ◽  
pp. 7029-7035 ◽  
Author(s):  
Rose Monnerat ◽  
Erica Martins ◽  
Paulo Queiroz ◽  
Sergio Ordúz ◽  
Gabriela Jaramillo ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Genetic Variability ◽  
Latin American ◽  
Spodoptera Frugiperda ◽  
Insect Pest ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cry Toxins ◽  
Geographical Regions ◽  
Insect Populations

ABSTRACT Bacillus thuringiensis strains isolated from Latin American soil samples that showed toxicity against three Spodoptera frugiperda populations from different geographical areas (Mexico, Colombia, and Brazil) were characterized on the basis of their insecticidal activity, crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parasporal crystals, plasmid profiles, and cry gene content. We found that the different S. frugiperda populations display different susceptibilities to the selected B. thuringiensis strains and also to pure preparations of Cry1B, Cry1C, and Cry1D toxins. Binding assays performed with pure toxin demonstrated that the differences in the toxin binding capacities of these insect populations correlated with the observed differences in susceptibility to the three Cry toxins analyzed. Finally, the genetic variability of the three insect populations was analyzed by random amplification of polymorphic DNA-PCR, which showed significant genetic diversity among the three S. frugiperda populations analyzed. The data presented here show that the genetic variability of S. frugiperda populations should be carefully considered in the development of insect pest control strategies, including the deployment of genetically modified maize in different geographical regions.

scholarly journals Crystalline protein profiling and cry gene detection in Bacillus thuringiensis strains isolated during epizootics in Cydia pomonella L.

2014 ◽  
Vol 51 (2) ◽  
pp. 83-92 ◽  
Author(s):  
Edyta Konecka ◽  
Jakub Baranek ◽  
Adam Kaznowski
Keyword(s):  
Bacillus Thuringiensis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cydia Pomonella ◽  
Sodium Dodecyl ◽  
Natural Populations ◽  
Protein Profiling ◽  
Gene Detection ◽  
Sds Page ◽  
Molecular Masses ◽  
Reference Strains

AbstractThe composition of Bacillus thuringiensis crystalline inclusions was characterized in 18 strains: 12 isolates were obtained from the intestinal tract of Cydia pomonella larvae during epizootics, 2 isolates were cultured from Leucoma salicis larvae taken from their natural populations, and 4 reference strains. The number and molecular mass of B. thuringiensis crystalline proteins (Cry and Cyt) was estimated by the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The crystals contained 1-8 proteins with molecular masses of 36-155 kDa. The toxin profiles differed both quantatively and qualitatively. The B. thuringiensis MPU B9 isolate had the highest number and diversity of Cry toxins. The analysis of crystal composition by SDS-PAGE was insufficient to detect groups and subgroups of Cry proteins. We identified 20 groups and 3 subgroups of Cry and Cyt crystalline toxins. Only one epizootic strain harboured cry25. In single reference strains, the cry1H, cry10 and cry25 genes were found. We did not find any correlation between the occurrence of cry genes and electrophoretic protein profiles of crystalline toxins.

scholarly journals Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

1982 ◽  
Vol 2 (4) ◽  
pp. 412-425 ◽  
Author(s):  
S I Reed ◽  
J Ferguson ◽  
J C Groppe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
In Vitro Mutagenesis ◽  
Coding Region ◽  
Loop Analysis ◽  
Partial Digestion ◽  
Polyadenylic Acid

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

scholarly journals Parasporal Body Formation via Overexpression of the Cry10Aa Toxin of Bacillus thuringiensis subsp. israelensis, and Cry10Aa-Cyt1Aa Synergism

2009 ◽  
Vol 75 (14) ◽  
pp. 4661-4667 ◽  
Author(s):  
Alejandro Hernández-Soto ◽  
M. Cristina Del Rincón-Castro ◽  
Ana M. Espinoza ◽  
Jorge E. Ibarra
Keyword(s):  
Bacillus Thuringiensis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Microbial Control ◽  
Control Agent ◽  
Mass Spectrometry Analysis ◽  
Parasporal Crystal ◽  
Spectrometry Analysis ◽  
Crystal Complex

ABSTRACT Bacillus thuringiensis subsp. israelensis is the most widely used microbial control agent against mosquitoes and blackflies. Its insecticidal success is based on an arsenal of toxins, such as Cry4A, Cry4B, Cry11A, and Cyt1A, harbored in the parasporal crystal of the bacterium. A fifth toxin, Cry10Aa, is synthesized at very low levels; previous attempts to clone and express Cry10Aa were limited, and no parasporal body was formed. By using a new strategy, the whole Cry10A operon was cloned in the pSTAB vector, where both open reading frames ORF1 and ORF2 (and the gap between the two) were located, under the control of the cyt1A operon and the STAB-SD stabilizer sequence characteristic of this vector. Once the acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis was transformed with this construct, parasporal bodies were observed by phase-contrast microscopy and transmission electron microscopy. Discrete, ca. 0.9-μm amorphous parasporal bodies were observed in the mature sporangia, which were readily purified by gradient centrifugation once autolysis had occurred. Pure parasporal bodies showed two major bands of ca. 68 and 56 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These bands were further characterized by N-terminal sequencing of tryptic fragments using matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis, which identified both bands as the products of ORF1 and ORF2, respectively. Bioassays against fourth-instar larvae of Aedes aegypti of spore-crystal complex and pure crystals of Cry10Aa gave estimated 50% lethal concentrations of 2,061 ng/ml and 239 ng/ml, respectively. Additionally, synergism was clearly detected between Cry10A and Cyt1A, as the synergistic levels (potentiation rates) were estimated at 13.3 for the mixture of Cyt1A crystals and Cry10Aa spore-crystal complex and 12.6 for the combination of Cyt1A and Cry10Aa pure crystals.

C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7

1994 ◽  
Vol 14 (9) ◽  
pp. 6164-6170
Author(s):  
P P Sadhale ◽  
N A Woychik
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rna Polymerase Iii ◽  
Sodium Dodecyl ◽  
Polymerase Iii ◽  
Conditional Mutant ◽  
5.8S Rrna ◽  
Cell Growth And Viability

We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

scholarly journals Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration

1977 ◽  
Vol 163 (2) ◽  
pp. 369-378 ◽  
Author(s):  
P R Dunkley ◽  
H Holmes ◽  
R Rodnight
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synaptic Membrane ◽  
Hydroxyl Groups ◽  
Phosphorylated Proteins

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

scholarly journals Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells

2005 ◽  
Vol 4 (11) ◽  
pp. 1951-1958 ◽  
Author(s):  
Felix D. Bastida-Corcuera ◽  
Cheryl Y. Okumura ◽  
Angie Colocoussi ◽  
Patricia J. Johnson
Keyword(s):  
Wheat Germ Agglutinin ◽  
Parent Strain ◽  
Trichomonas Vaginalis ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Chemical Mutagenesis ◽  
Reduced Adherence

ABSTRACT The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.

scholarly journals Proteolytic Processing of Sapovirus ORF1 Polyprotein

2005 ◽  
Vol 79 (12) ◽  
pp. 7283-7290 ◽  
Author(s):  
Tomoichiro Oka ◽  
Kazuhiko Katayama ◽  
Satoko Ogawa ◽  
Grant S. Hansman ◽  
Tsutomu Kageyama ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Open Reading Frames ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Translation System ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Capsid Protein Vp1

ABSTRACT The genome of Sapovirus (SaV), a causative agent of gastroenteritis in humans and swine, contains either two or three open reading frames (ORFs). Functional motifs characteristic to the 2C-like NTPase (NTPase), VPg, 3C-like protease (Pro), 3D-like RNA-dependent RNA polymerase (Pol), and capsid protein (VP1) are encoded in the ORF1 polyprotein, which is afterwards cleaved into the nonstructural and structural proteins. We recently determined the complete genome sequence of a novel human SaV strain, Mc10, which has two ORFs. To investigate the proteolytic cleavage of SaV ORF1 and the function of protease on the cleavage, both full-length and truncated forms of the ORF1 polyprotein either with or without mutation in 1171Cys to Ala of the GDCG motif were expressed in an in vitro coupled transcription-translation system. The translation products were analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by immunoprecipitation with region-specific antibodies. The ORF1 polyprotein was processed into at least 10 major proteins: p11, p28, p35, p32, p14, p70, p60, p66, p46, and p120. Seven of these products were arranged in the following order: NH2-p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)-COOH. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of other heretofore known members of the family Caliciviridae, especially to rabbit hemorrhagic disease virus, a member of the genus Lagovirus.

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